Nucleic acid is an important analysis tool when identifying a disease state. DNA biomarkers (e.g. single nucleotide polymorphism (SNP), mutation, and DNA methylation) offer important clues to help researchers look for the causes of cancer and provide great opportunities to diagnose and monitor disease status during the early stages of diseases, as well as for prognosis and surveillance. Because of the extremely low physiological concentration of DNA compared to other components such as proteins (i.e. tens of nanograms of DNA versus tens of micrograms of protein in a microliter of whole blood), efficient extraction and pre-concentration of DNA from clinical samples is critical for the subsequent downstream processes such as amplification and detection. When it comes to methylated DNA this problem is magnified.
DNA methylation plays a crucial role in the regulation of gene expression and chromatin organization within normal eukaryotic cells. DNA methylation occurs by covalent addition of a methyl group at the 5 carbon of the cytosine ring, resulting in 5-methylcytosine. These methyl groups project into the major groove of DNA and effectively inhibit transcription. In mammalian DNA, 5-methylcytosine is found in approximately 4% of genomic DNA, primarily at cytosine-guanosine dinucleotides (CpGs). Such CpG sites occur at lower than expected frequencies throughout the human genome but are found more frequently at small stretches of DNA called CpG islands. These islands are typically found in or near promoter regions of genes, where transcription is initiated. In contrast to the bulk of genomic DNA, in which most CpG sites are heavily methylated, CpG islands in germ-line tissue and promoters of normal somatic cells remain unmethylated, allowing gene expression to occur. DNA methylation is mediated by a family of highly related DNA methyltransferase enzymes (DNMT), which transfer a methyl group from S-adenosyl-L_methionine to cytosines in CpG dinucleotides. The methyl-cytosines established by the DNMTs serve as binding sites for the methyl-CpG binding domain (MBD) proteins MeCP2, MBD (S. B. Baylin, DNA methylation and gene silencing in cancer. Nature Clin. Prac. Oncol. 2 (2005) 4-11.; M. T. McCabe, et al., Cancer DNA methylation: Molecular mechanisms and clinical implications. Clin. Cancer Res. 15 (2009) 3927-3937.; M. Wielscher, et al. Methyl-binding domain protein-based DNA isolation from human blood serum combines DNA analyses and serum-autoantibody testing. BMC Clin. Pathol. 11 (2011) 11-20.; and B. R. Cipriany, et al. Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel. Proc. Nat. Acad. Sci USA. 109 (2012) 8477-8482. Through interactions with histone deacetylases, histone methyltransferases, and ATP-dependent chromatin remodeling enzymes, the MBDs translate methylated DNA into a compacted chromatin environment that is repressive for transcription. Especially, MBD is the methyl CpG binding domain of the MeCP2 protein, which binds symmetrically methylated CpGs in any sequence context, and is involved in mediating methylation dependent transcriptional repression. Although there is a strong evidence that MeCP2 binds exclusively methylated DNA fragments in vivo, a DNA methylation-independent binding activity of MeCP2 in vitro was also described in concordant literature, which makes it suitable for general in vitro DNA analysis [S. B. Baylin; McCabe, et al.; M. Wielscher, et al.; and B. R. Cipriany, et al.].
DNA methylation causes silencing of expression of tumor suppressor genes in human cancers. An ever increasing body of work within the field of epigenomics is strengthening the linkage between the hypermethylation of key nucleotide sequences and the advent of many different cancers. DNA methylation patterns in human cancer cells are considerably distorted. Typically, cancer cells exhibit hypomethylation of intergenic regions that normally comprise the majority of a cell's methyl-cytosine content. Consequently, transposable elements may become active and contribute to the genomic instability observed in cancer cells. Simultaneously, cancer cells exhibit hypermethylation within the promoter regions of many CpG island-associated tumor suppressor genes. As a result, these regulatory genes are transcriptionally silenced resulting in a loss of function. Thus, through the effects of both hypo- and hypermethylation, DNA methylation significantly affects the genomic landscape of cancer cells, potentially to an even greater extent than coding region mutations, which are relatively rare [S. B. Baylin; McCabe, et al.; M. Wielscher, et al.; and B. R. Cipriany, et al]. DNA methylation is of great important for cancer research and clinics, since it enables earlier cancer diagnosis prior to the point of metastasis. An example is RARβ, a thyroid-steroid hormone receptor that controls the growth of many cell types by regulating gene expression. Methylation of RARβ has been reported in breast, lung, and bladder cancers.
The recent development of several genome-scale methylation screening technologies has considerably expanded our understanding of DNA methylation patterns, both in normal and cancerous cells. Particularly, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus. The chemical modification of cytosine to uracil by bisulfite treatment has provided another method for the study of DNA methylation that avoids the use of restriction enzymes. However, these methods are technically rather difficult and labor-intensive, and, without cloning of the amplified products, the technique is less sensitive than Southern analysis, requiring—25% of the alleles to be methylated for detection. Therefore, the isolation of the methylated DNA from human genomic DNA is an important step for improving of DNA methylation analysis in cancer, but that is still challenging [J. G. Herman, J. R. et al., Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Nat. Acad. Sci USA. 93 (1996) 9821-9826.; S. Pan, et al., Double recognition of oligonucleotide and protein in the detection of DNA methylation with surface Plasmon resonance biosensors. Biosens. Bioelectron. 26 (2010) 850-853.; and J. D. Suter, et al., Label-free DNA methylation analysis using opto-fluidic ring resonators. Biosens. Bioelectron. 26 (2010) 1016-102].
In solution phase methods, for the isolation of the methylated DNA from genomic DNA, up to now, recombinant MBD protein, which is available upon overexpression of the cloned His-tagged protein in E. coli, has been predominantly used for DNA methylation analyses. The MBD protein has been preferably applied being immobilized in an affinity chromatography like manner with NaCl gradient elution steps to isolate methylated DNA for PCR and gel analyses in solution phase. According to commercialized protocol from companies, the MBD protein attached to Ni-Sepharose or Magnetic beads for affinity based DNA purification that enables the simultaneous analyses of the methylated DNA. MBD isolated the DNA has been found particularly suitable for DNA methylation analyses (FIG. 1, Black_line).
However, the previous studies for the detection of DNA methylation based label-free biosensor without bisulfite modification have been so far demonstrated only with synthetic oligonucleotides. The direct detection of native methylated DNAs in genomic DNA in bodily fluids such as blood, urine, or saliva would be difficult due to their extremely low concentration. The number of a specific gene in total DNA is extremely low. For instance, Su et al. have reported that about 2 copies of mutated tumor Kristin-ras DNA can be found in 50-200 μL of urine or blood samples of cancer patients [Y. H. Su, et al., Block, Detection of mutated K-ras DNA in urine, plasma, serum of patients with colorectal carcinoma or adenomatous polyps, Annals of the New York Academy of Sciences 1137 (2008) 197-206.]. The sensitivity of the reported label-free biosensors is not good enough to detect such a low concentration of native DNA biomarkers. Therefore, these label-free techniques are inadequate to be used as in vitro diagnostic (IVD) device without amplification of target DNAs.
It has been recently reported that highly sensitive silicon-based microring resonators were used to detect biomolecules (e.g., protein, methylated DNA, nucleic acids) by monitoring a shift in the resonant wavelength. Optical refractive index (RI) sensors are extensively investigated for a number of applications and play a prominent role in biochemical analysis. Among the existing biochemical RI sensors, those based on integrated optical waveguides are of great interest because of their high sensitivity, small size, and high scale integration. Recently, RI sensors based on slot waveguide have attracted significant interest due to slot waveguide's remarkable property to provide high optical intensity in a sub-wavelength-size low refractive index region (slot region) sandwiched between two high refractive index strips. Using the slot as sensing region, larger light-analyte interaction, and hence higher sensitivity, can be obtained as compared to a conventional waveguide. The sensing light is concentrated close to the surface by an evanescent field undergoing exponential decay with a characteristic decay length of up to few hundred nanometers. Thus, the refractive index is affected by the binding of the analyte with immobilized capturing ligand within the decay length. Silicon microring resonators are refractive-index-based optical sensors that provide highly sensitive, label-free, real-time multiplexed detection of biomolecules near the sensor surface. Furthermore, the devices are fabricated by using standard CMOS technology, which ensures low cost and scale-up capability. The methods require time consuming steps of immobilizing probes on the surface of the device by highly skilled personnel.
The object of the invention is to ameliorate at least some of the above mentioned difficulties.